Abstract Pathogenic bacteria within a mammalian host are bombarded by intercellular, interspecies, and cross-kingdom signaling molecules and metabolites. For Salmonella enterica, multiple signals, including pH, bile, and short chain fatty acids, regulate Salmonella Pathogenicity Island I (SPI-1) to trigger invasion and inflammation in the gut. Recently, our lab demonstrated that the methionine-derived metabolite methylthioadenosine (MTA) is a previously unrecognized signal produced by the host and pathogen that suppresses SPI-1, flagellar motility, and in vivo virulence. These conclusions were drawn from experiments treating S. Typhimurium with MTA and by characterizing a mutant with increased endogenous MTA. This mutant lacks the master repressor of the methionine metabolism pathway, metJ. In addition to MTA?s effect on S. Typhimurium, MTA also modulates host responses and is released into plasma during S. Typhimurium infection in mice and during sepsis in humans. Thus, MTA can suppress S. Typhimurium virulence and its levels in the host undergo dramatic shifts during infection. However, it is unknown if and how host- or microbiota-produced MTA signals to S. Typhimurium at the crucial initial site of this host-pathogen conflict, the intestinal lumen. We hypothesize that MTA in the intestine is dynamically regulated by host, commensal microbiota, and pathogen, and that shifts in MTA levels are sensed by Salmonella to regulate virulence gene expression. Therefore, the first goal of this project is to characterize contributions and consequences of host- and microbial-derived MTA during S. Typhimurium infection. MTA levels in gut contents and tissue will be measured from germ-free and microbiome reconstituted mice at baseline and with S. Typhimurium infection. Furthermore, we will manipulate host MTA levels through exogenous MTA and the use of an inhibitor that specifically blocks host MTA metabolism. The second goal is to elucidate mechanisms of MTA suppression of S. Typhimurium virulence. We will test whether MTA modulates regulators of SPI-1 expression and if MTA facilitates broader gene expression changes through regulation of methylation. Completion of these aims will identify whether MTA acts as an interspecies signal in the gut and identify how MTA suppresses virulence. This work could improve clinical outcomes by demonstrating that existing methionine metabolism inhibitors can suppress S. Typhimurium virulence.